Mycobacterial diseases, predominantly tuberculosis, chronically infect billions and kill nearly two million people annually. This global crisis is exacerbated by inadequate treatments: the BCG vaccine is of questionable efficacy, and the incidence of multi-drug and extremely-drug resistant strains of M. tuberculosis is increasing alarmingly. We will apply genomics and high throughput technologies to develop a Mycobacterial Systems Resource (MSR) that will dramatically accelerate research throughout the entire mycobacterial community. The MSR will be freely available and will provide a comprehensive, integrated genomic and visual summation of biological processes common to all mycobacteria that can be immediately applied to further our understanding of mycobacterial diseases. Insights gained from the MSR will generate a plethora of new biological and comparative tools and identify many new targets suitable for the development of novel anti- mycobacterial therapeutics. Thus, the MSR will stimulate the mycobacterial research community, and facilitate the translation of basic discoveries to combat species that cause an enormous global health burden. These goals will be achieved by taking advantage of the combined expertise and cutting-edge technologies available to the researchers assembled to form this multi-disciplinary, multi-institutional team. Pathogenic mycobacteria are extremely slow-growing and hazardous to work with, making them inappropriate for high-throughput genomic analyses. We will obviate this problem by using a closely related species, M. smegmatis, which is a fast-growing, non-pathogenic mycobacterium that is the acknowledged genetically tractable model Mycobacterium. The focus of the MSR will be ~1,300 M. smegmatis coding and non-coding genes that are highly conserved between members of the M. tuberculosis complex, M. avium, M. ulcerans and M. leprae. These highly conserved genes will mediate fundamental cellular processes and therefore their analysis will be applicable to all mycobacterial species. The MSR will have two components: physical and electronic database (or eSource). The physical resource will consist of a targeted deletion or knockdown of ~1,300 highly conserved coding and non-coding mycobacterial genes, and a collection of expression vectors with the coding genes fused to functional tags to facilitate future analyses. The eSource will describe a wide variety of initial phenotypic characterizations of the strains and plasmids created in the physical resource. These include microscopic analyses of protein localization and cellular morphology, identification of genes associated with sensitivity to anti-mycobacterial drugs, and genes influencing the cell's surface-environment interface. The data generated from these studies will be assembled and integrated on the online Tuberculosis Database (TBDB) and will be freely available to the scientific community. In summary, the MSR will provide unprecedented insight into mycobacterial biology and will serve as a launch pad for future studies by researchers spanning the mycobacterial community.